The present invention relates to transgenic nonhuman animals wherein an H2-O (the mouse equivalent of human HLA-DO), gene is altered, producing an animal lacking functional H2-O protein.
Immediately after synthesis in the endoplasmic reticulum (ER), major histocompatibility complex (MHC) class II molecules associate with the invariant chain (Ii). Ii inhibits binding of peptides and nascent proteins to class II molecules in the ER and directs Ii-class II complexes to the endosomal system where binding of antigenic peptides occur (Wolf and Ploegh, 1995). Ii is degraded by proteolysis, but complete removal of class II-associated invariant chain peptides (CLIP) requires the catalytic function of HLA-DM (DM), a resident of the endosomal/lysosomal system which is structurally related to class II molecules (Fling et al., 1994; Karlsson et al., 1994; Morris et al., 1994; Sanderson et al., 1994). The absence of DM (or H2-M, the equivalent mouse molecule) leads to the accumulation of CLIP-containing class II molecules and decreased loading of antigenic peptides (Fung-Leung et al., 1996; Martin et al., 1996; Mellins et al., 1990; Miyazaki et al., 1996). In vitro, DM is sufficient to release CLIP peptides from class II molecules, but this effect is not restricted to CLIP (Kropshofer et al., 1997; Sloan et al., 1995; Weber et al., 1996). Thus, the dissociation rate of any peptide from class II molecules appears to be increased in the presence of DM, but remains proportional to the intrinsic dissociation rate of the peptide (Kropshofer et al., 1997; Weber et al., 1996). Whether DM-mediated release of peptides other than CLIP is important also in vivo is presently unknown. In B cells the majority of DM is associated with HLA-DO (DO) (Liljedahl et al., 1996), and a recent report has shown that association with DO inhibits the ability of DM to release CLIP, both in vitro and in transfected cells (Denzin et al., 1997). The physiological relevance of this effect is unclear however, since CLIP-containing class II molecules are not particularly prominent on B cells, the main, if not exclusive, cell type expressing DO (Douek and Altmann, 1997; Karlsson et al., 1991; Liljedahl et al., 1996; Tonelle et al., 1985; Wake and Flavell, 1985).
In contrast to dendritic cells, which are comitted to antigen presentation irrespective of antigen specificity (Cella et al., 1997), B cells are themselves antigen specific, but usually require T cell help in order to mature into antibody-secreting plasma cells (Vitetta et al., 1991). The antigens presented by B cells are mainly internalized by their immunoglobulin surface receptors (mIg) (Lanzavecchia, 1985; Rock et al., 1984) and receptor-independent antigen presentation by B cells, though well studied in vitro, is relatively inefficient. How B cells focus antigen presentation to antigens internalized by the B cell receptor is unclear, since increased antigen capture can only partly explain this phenomenon (Watts, 1997). It has been suggested that efficient B cell receptor-mediated antigen presentation may require specialized loading compartments (Mitchell et al., 1995; Watts, 1997) and several groups have described class II-rich intracellular compartments (Amigorena et al., 1994; Peters et al., 1991; Tulp et al., 1994; West et al., 1994). These compertments are not restricted to B cells, however (Calafat et al., 1994; Kleijmeer et al., 1994) and their functional importance is unclear. Though the general machinery for antigen processing is likely to be the same in different antigen presenting cells (APCs), it is still possible that cell-type specific differences in antigen processing may contribute to the efficient presentation of antigens internalized by mIg.
The present invention provides mice lacking H2-Oa to evaluate the importance of H2-O/DO for peptide loading and antigen presentation. Cells from H2-Oa-deficient mice were found to have normal levels of class II expression, and the density of H2-Ab-CLIP complexes at the cell surface was the same as in wild-type controls. However, B cells from H2-Oa deficient mice were found to have changed capacity to present protein antigens when compared to B cells from wild-type mice, showing that the absence of H2-O does modify the peptide content of class II molecules at the cell surface, either qualitatively or quantitatively. In older H2-Oa-deficient mice serum levels of immunoglobulin G1 (IgG1) were elevated, further showing that T-B cell interaction may be changed in these mice. Analysis of DO function in vitro, using recombinant molecules, confirmed the finding by Denzin et al. that DO inhibits DM function, but showed that the inhibition is decreased at acidic pH, suggesting that peptide loading may be favoured in acidic compartments (probably lysosomes) in the presence of DO.
To understand the functional role of H2-O in different cell types, mice that do not express the functional H2-O were generated by homologous recombination (HR) in embryonic stem (ES) cells and are disclosed herein. These mice provide a valuable animal model to understand the function of H2-O and to evaluate the therapeutic effects of drugs that modulate the function or the expression of H2-O equivalents in human cells.